Angiogenesis is the generation of new blood vessels into a tissue or organ. Under normal physiological conditions, humans and animals undergo angiogenesis only in very specific restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonal development, and formation of the corpus luteum, endometrium and placenta.
Angiogenesis is controlled through a highly regulated system of angiogenic stimulators and inhibitors. The control of angiogenesis has been found to be altered in certain disease states and, in many cases, pathological damage associated with the diseases related to uncontrolled angiogenesis. Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. Endothelial cells, lining the lumen of blood vessels then protrude through the basement membrane. Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane. The migrating cells form a "sprout" off the parent blood vessel where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating a new blood vessel.
Persistent, unregulated angiogenesis occurs in a multiplicity of disease states, tumor metastases, and abnormal growth by endothelial cells. The diverse pathological disease states in which unregulated angiogenesis is present have been grouped together as angiogenic-dependent or angiogenic-associated diseases.
One example of a disease mediated by angiogenesis is ocular neovascular disease. This disease is characterized by invasion of new blood vessels into the structures of the eye, such as the retina or cornea. It is the most common cause of blindness and is involved in approximately twenty eye diseases. In age-related macular degeneration, the associated visual problems are caused by an ingrowth of choroidal capillaries through defect in Bruch's membrane with proliferation of fibrovascular tissue beneath the retinal pigment epithelium. Angiogenic damage is also associated with diabetic retinopathy, retinopathy of prematurity, corneal graph rejection, neovascular glaucoma, and retrolental fibroplasia. Other diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens disease, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infection, Herpes zoster infections, protozoan infections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegener's sarcoidosis, scleritis, Stevens-Johnson's disease, pemphigoid, and radial keratotomy.
Diseases associated with retinal/choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoidosis, syphilis, pseudoxanthoma elasticum, Paget's disease, vein occlusion, artery occlusion, carotid obstructive disease, chronic uveitis/vitritis, Mycobacteria infections, lyme's disease, systemic lupus erythematosis, retinopathy of prematurity, Eale's disease, Behcet's disease, infections causing a retinitis or choroiditis, presumed ocular histoplasmosis, Best's disease, myopia, optic pits, Stargardt's disease, pars planitis, chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis, trauma and post-laser complications. Other eye-related diseases include, but are not limited to, diseases associated with rubeosis (neovascularization of the angle) and diseases caused by the abnormal proliferation of fibrovascular or fibrous tissue, including all forms of prolific vitreoretinopathy.
Another angiogenesis associated disease is rheumatoid arthritis. The blood vessels in the synovial lining of the joints undergo angiogenesis. In addition to forming new vascular networks, the endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction. Angiogenesis may also play a role in osteoarthritis. The activation of the chondrocytes by angiogenic-related factors contributes to the destruction of the joint. At a later stage, the angiogenic factors promote new bone growth. Therapeutic intervention that prevents the bone destruction could halt the progress of the disease and provide relief for persons suffering with arthritis.
Chronic inflammation may also involve pathological angiogenesis. Such diseases as ulcerative colitis and Crohn's disease show histological changes with the ingrowth of new blood vessels and the inflamed tissues. Bartonelosis, a bacterial infection found in South America, can result in a chronic stage that is characterized by proliferation of vascular endothelial cells. Another pathological role associated with angiogenesis is found in atherosclerosis. The plaques formed within the lumen of blood vessels have been shown to have angiogenic stimulatory activity.
The hypothesis that tumor growth is angiogenesis-dependent was first proposed in 1971. (Folkman, New Eng. J. Med., 285:1182-86 (1971)) In its simplest terms, this hyposthesis states: "Once tumor `take` has occurred, every increase in tumor cell population must be preceded by an increase in new capillaries converging on the tumor." Tumor `tak` is currently understood to indicate a prevascular phase of tumor growth in which a population of tumor cells occupying a few cubic millimeters volume, and not exceeding a few million cells, can survive on existing host microvessels. Expansion of tumor volume beyond this phase requires the induction of new capillary blood vessels. For example, pulmonary micrometastases in the early prevascular phase in mice would be undetectable except by high power microscopy on histological sections.
Examples of the indirect evidence which support this concept include:
(1) The growth rate of tumors implanted in subcutaneous transparent chambers in mice is slow and linear before neovascularization, and rapid and nearly exponential after neovascularization. (Algire, et al., J. Nat. Cancer Inst., 6:73-85 (1945)).
(2) Tumors grown in isolated perfused organs where blood vessels do not proliferate are limited to 1-2 mm.sup.3 but expand rapidly to &gt;1000 times this volume when they are transplanted to mice and become neovascularized. (Folkman, et al., Annals of Surgery, 164:491-502 (1966)).
(3) Tumor growth in the avascular cornea proceeds slowly and at a linear rate, but switches to exponential growth after neovascularization. (Gimbrone, Jr., et al., J. Nat. Cancer Inst., 52:421-27 (1974)).
(4) Tumors suspended in the aqueous fluid of the anterior chamber of the rabbit eye remain viable, avascular, and limited in size to &lt;1 mm.sup.3. Once they are implanted on the iris vascular bed, they become neovascularized and grow rapidly, reaching 16,000 times their original volume within 2 weeks. (Gimbrone, Jr., et al., J. Exp. Med., 136:261-76).
(5) When tumors are implanted on the chick embryo chorioallantoic membrane, they grow slowly during an avascular phase of &gt;72 hours, but do not exceed a mean diameter of 0.93+0.29 mm. Rapid tumor expansion occurs within 24 hours after the onset of neovascularization, and by day 7 these vascularized tumors reach a mean diameter of 8.0+2.5 mm. (Knighton, British J. Cancer, 35:347-56 (1977)).
(6) Vascular casts of metastases in the rabbit liver reveal heterogeneity in size of the metastases, but show a relatively uniform cut-off point for the size at which vascularization is present. Tumors are generally avascular up to 1 mm in diameter, but are neovascularized beyond that diameter. (Lien, et al., Surgery, 68:334-40 (1970)).
(7) In transgenic mice which develop carcinomas in the beta cells of the pancreatic islets, pre-vascular hyperplastic islets are limited in size to &lt;1 mm. At 6-7 weeks of age, 4-10% of the islets become neovascularized, and from these islets arise large vascularized tumors of more than 1000 times the volume of the pre-vascular islets. (Folkman, et al., Nature, 339:58-61 (1989)).
(8) A specific antibody against VEGF (vascular endothelial growth factor) reduces microvessel density and causes "significant or dramatic" inhibition of growth of three human tumors which rely on VEGF as their sole mediator of angiogenesis (in nude mice). The antibody does not inhibit growth of the tumor cells in vitro. (Kim, et al., Nature, 362:841-44 (1993)).
(9) Anti-bFGF monoclonal antibody causes 70% inhibition of growth of a mouse tumor which is dependent upon secretion of bFGF as its only mediator of angiogenesis. The antibody does not inhibit growth of the tumor cells in vitro. (Hori, et al., Cancer Res., 51:6180-84 (1991)).
(10) Intraperitoneal injection of bFGF enhances growth of a primary tumor and its metastases by stimulating growth of capillary endothelial cells in the tumor. The tumor cells themselves lack receptors for bFGF, and bFGF is not a mitogen for the tumors cells in vitro. (Gross, et al., Proc. Am. Assoc. Cancer Res., 31:79 (1990)).
(11) A specific angiogenesis inhibitor (AGM-1470) inhibits tumor growth and metastases in vivo, but is much less active in inhibiting tumor cell proliferation in vitro. It inhibits vascular endothelial cell proliferation half-maximally at 4 logs lower concentration than it inhibits tumor cell proliferation. (Ingber, et al., Nature, 48:555-57 (1990)). There is also indirect clinical evidence that tumor growth is angiogenesis dependent.
(12) Human retinoblastomas that are metastatic to the vitreous develop into avascular spheroids which are restricted to less than 1 mm.sup.3 despite the fact that they are viable and incorporate .sup.3 H-thymidine (when removed from an enucleated eye and analyzed in vitro).
(13) Carcinoma of the ovary metastasizes to the peritoneal membrane as tiny avascular white seeds (1-3 mm.sup.3). These implants rarely grow larger until one or more of them becomes neovascularized.
(14) Intensity of neovascularization in breast cancer (Weidner, et al., New Eng. J. Med., 324:1-8 (1991); Weidner, et al., J Nat. Cancer Inst., 84:1875-87 (1992)) and in prostate cancer (Weidner, et al., Am. J Pathol., 143(2):401-09 (1993)) correlates highly with risk of future metastasis.
(15) Metastasis from human cutaneous melanoma is rare prior to neovascularization. The onset of neovascularization leads to increased thickness of the lesion and an increased risk of metastasis. (Srivastava, et al., Am. J. Pathol., 133:419-23 (1988)).
(16) In bladder cancer, the urinary level of an angiogenic protein, bFGF, is a more sensitive indicator of status and extent of disease than is cytology. (Nguyen, et al., J. Nat. Cancer Inst., 85:241-42 (1993)).
Thus, it is clear that angiogenesis plays a major role in the metastasis of cancer. If this angiogenic activity could be repressed or eliminated, then the tumor, although present, would not grow. In the disease state, prevention of angiogenesis could avert the damage caused by the invasion of the new microvascular system. Therapies directed at control of the angiogenic processes could lead to the abrogation or mitigation of these diseases.
Angiogenesis has been associated with a number of different types of cancer, including solid tumors and blood-borne tumors. Solid tumors with which angiogenesis has been associated include, but are not limited to, rhabdomyosarcomas, retinoblastoma, Ewing's sarcoma, neuroblastoma, and osteosarcoma. Angiogenesis is also associated with blood-borne tumors, such as leukemias, any of various acute or chronic neoplastic diseases of the bone marrow in which unrestrained proliferation of white blood cells occurs, usually accompanied by anemia, impaired blood clotting, and enlargement of the lymph nodes, liver and spleen. It is believed to that angiogenesis plays a role in the abnormalities in the bone marrow that give rise to leukmia-like tumors.
One of the most frequent angiogenic diseases of childhood is the hemangioma. Hemangioma is a tumor composed of newly-formed blood vessels. In most cases the tumors are benign and regress without intervention. In more severe cases, the tumors progress to large cavernous and infiltrative forms and create clinical complications. Systemic forms of hemangiomas, hemangiomatoses, have a high mortality rate. Therapy-resistant hemangiomas exist that cannot be treated with therapeutics currently in use.
Angiogenesis is also responsible for damage found in heredity diseases such as Osler-Weber-Rendu disease, or heredity hemorrhagic telangiectasia. This is an inherited disease characterized by multiple small angiomas, tumors of blood or lymph vessels. The angiomas are found in the skin and mucous membranes, often accompanied by epitaxis (nose bleeds) or gastrointestinal bleeding and sometimes with pulmonary or hepatitic arteriovenous fistula.
What is needed, therefore, is a composition and method which can inhibit angiogenesis. What is also needed is a composition and method which can inhibit the unwanted growth of blood vessels, especially in tumors.
Angiogenesis is also involved in normal physiological processes, such as reproduction and wound healing. Angiogenesis is an important step in ovulation and also in implantation of the blastula after fertilization. Prevention of angiogenesis could be used to induce amenorrhea, to block ovulation, or to prevent implantation by the blastula.
In wound healing, excessive repair or fibroplasia can be a detrimental side effect of surgical procedures and may be caused or exacerbated by angiogenesis. Adhesions are a frequent complication of surgery and lead to problems such as small bowel obstruction.
Several compounds have been used to inhibit angiogenesis. Taylor, et al. (Nature, 297:307 (1982)) have used protamine to inhibit angiogenesis. The toxicity of protamine limits its practical use as a therapeutic. Folkman, et al. (Science, 221:719 (1983), and U.S. Pat. Nos. 5,001,116 and 4,994,443) have disclosed the use of heparin and steroids to control angiogenesis. Steroids, such as tetrahydrocortisol, which lack gluccocorticoid and mineralocorticoid activity, have been found to be anglogenic inhibitors.
Other factors found endogenously in animals, such as a 4 kDa glycoprotein from bovine vitreous humor and a cartilage derived factor, have been used to inhibit angiogenesis. Cellular factors, such as interferon, inhibit angiogenesis. For example, interferon alpha or human interferon beta have been shown to inhibit tumor-induced angiogenesis in mouse dermis stimulated by human neoplastic cells. Interferon beta is also a potent inhibitor of angiogenesis induced by allogeneic spleen cells. (Sidky, et al., Cancer Res., 47:5155-61(1987)). Human recombinant interferon (alpha/A) was reported to be successfully used in the treatment of pulmonary hemangiomatosis, an angiogenesis-induced disease. (White, et al., New Eng. J. Med., 320:1197-1200 (1989)).
Other agents which have been used to inhibit angiogenesis include ascorbic acid ethers and related compounds. (Japanese Kokai Tokkyo Koho No.58-13 (1978)). Sulfated polysaccharide DS 4152 also inhibits angiogenesis. (Japanese Kokai Tokkyo Koho No. 63-119500).
The above compounds lack adequate potency or are too toxic for practical use. Thus, methods and compositions are needed that are easily administered and capable of inhibiting angiogenesis.
Cytochalasins are secondary metabolites of mold and fungi. They are classified into two groups, Ascomycotina and Deuteromycotina. Four other types of compounds are associated with cytochalasins due to their similarity in chemical structure and activity. These are Chaetoglobosins (Chaetomium sp.), Aspochalasins (Aspergillus sp.), Zygosporins (Zygosporum sp.), and Phomins (Phoma sp.). The cytochalasins include fifteen compounds named from A to M (e.g., cytochalasin A); the chaetoglobulins include 13 compounds named from A to K; the aspochalasins include four compounds named from A to D. There are five zygosporins and five phomins which are each associated with a different cytochalasin. There are also many known derivatives of each of the various cytochalasins, for example, 17-hydroxycytochalasins, 19,20-dihydrocytochalasins, and substituted cytochalasins. (Cytochalasins: Biochemical and Aspects, S. W. Tannenbaum, ed., North Holland Pub. Co., 15, 18, 320 (1978)). Reduction products of cytochalasins are also known. (Steyn, et al., J. Chem. Soc., Perkin Trans 1, 541-44 (1982)).
Structurally, cytochalasins contain a highly substituted hydrogenated isoindolinone ring system, called cytochalasan, to which a macrocyclic ring is fused. The macrocyclic ring varies from 11 to 14 atoms in size and is either a carbocyclic ring, a lactone, or a cyclic carbonate. The major structural differences amongst cytochalasins, chaetoglobosins, and aspochalasins are a phenyl ring, indolyl group, and isopropyl group respectively at the C-10 position.
Cytochalasins can be produced by fermentation. For example, Adridge, et al. (J. Chem. Soc., C: 1667-76 (1967)) have suggested that cytochalasins A and B can be produced from fermentation on Raulin's medium. Other cytochalasins can be produced from KC medium with shredded wheat. (Springer, et al., Tet. Lett., 1355-58 (1976); Zabel, et al., Appl. Environ. Microbiol., 37:208-17 (1979); Cutler, et al., J. Agric. Food Chem., 28: 139-42 (1980); Probst and Tamm, Helv. Chem. Acta., 64(7): 2056-64 (1981)) Some cytochalasins have also been produced by shake culture using glucose, soybean cake, KH.sub.2 PO.sub.4 and corn steep liquor medium. (Sekita, et al., Tet. Lett., 1351-54 (1976)).
Several biosynthetic pathways have been used to generate cytochalasins. For example, cytochalasin D has been synthesized from Zygosporium masonii using .sup.13 C and .sup.14 C labelled sodium acetate, propionate, and malonate. This process uses the acetate malonate pathway to generate a C-16 polyketide moiety in which eight acetate units are linked head to tail. The polyketide is then incorporated with L-phenylalanine, followed by condensation to form a 5-membered lactum. The lactum undergoes reduction and dehydration, followed by Diels-Alder cyclization to give a cytochalasin. The carbonate and lactone ring system can be formed, for example, by Bayer-Villiger type oxidation of the macrocycle. (Binder, et al., J. Chem. Soc. Perkin Trans., 1: 1146-47 (1973); Lebet and Tamm, Helv. Chem. Acta., 57: 1785-1801 (1974); Vederas and Tamm, Helv. Chim. Acta., 59: 558-66 (1976); Vederas, et al., Helv. Chim. Acta, 58, 1886-98 (1975); and Wyss, et al., Helv. Chim. Acta, 63: 1538-41 (1980)).
Another method for producing cytochalasins is Kolbe coupling. For example, cytochalasin B has been synthesized from (+) citronellol and (+) malic acid derivatives. (Stork, et al., J. Am. Chem. Soc., 100: 7775-77 (1978)). Vedejs and Reid have also described the synthesis of zygosporin G. (Vedejs and Reid, J. Am. Chem. Soc., 106: 4617-18 (1984)). Additionally, a number of researchers have described the partial synthesis of cytochalasins, such as formation of the isoindolinone unit and cycloundecaconone. (Kim and Weinreb, Tet. Lett., 20: 576-82 (1979); Owen and Raphael, J. Chem. Soc. Perkin Trans., 1: 1504-07 (1978).
Cytochalasins are capable of eliciting and moderating several cellular activities, such as enucleation of cells, inhibition of cell motility, and interference with cytoplasmic cleavage. Cytochalasins also affect the transportation of certain biochemicals across the cell memberane.
In a study by Carter (Nature, 293: 302-5 (1967)), cytochalasins displaced the nuclei from the cytoplasm of cultured L929 cells without affecting cell variability. This response is dose and time dependent. At lower doses and incubation times, the effect can be reversed by transferring the cells to medium which does not contain cytochalasin. At higher doses and incubatoin times, the L929 cells undergo enucleation.
The mechanism for enucleation is not known; however, several hypotheses have been proposed. Poste and Lyon (Cytochalasins: Biochemical and Cell Biological Aspect, S. W. Tannenbaum, ed., 161-89 (1978)) suggest that enucleation occurs due to depolymerization of cortical microfilaments, along with an increase in hydrostatic pressure in the cytoplasm. Bhisey, et al. (Exp. Cell Res., 95: 376-84 (1975)) suggest that enucleation occurs due to active contraction of cytoplasm. Their studies showed a number of morphological changes in cell structure when the cells were treated with cytochalasin B which indicates that enucleation is an active, rather than passive, phenomenon.
Cytochalasins exhibit a number of cytotoxic and teratogenetic effects. These include mutinucleation, inhibition of fertilization, teratogenic effects, and chromosomal abnormalities. Cytochalasins have been shown to depolymerize microfilaments in the contractile ring during telophase, resulting in nuclear division not followed by cytokinesis. Thus, multimucleated cells are produced. (Aubin, et al., Exp. Cell Res., 136: 63-79 (1981)). The affects of cytochalasin on microfilaments also results in inhibition of DNA synthesis (O'Neil, J. Cell Physio., 101: 201-17 (1980)) and inhbition of cytokinesis. (Cytochalasins: Biochemical and Cell Biological Aspect, S. W. Tannenbaum, ed., North Holland Pub. Co., 217-55 (1978); O'Neil and Renzetti, Cancer Res., 43: 521-28 (1983); Maness and Walsh, Cell, 30: 253-62 (1982)). Further, inhibition of microfilaments inhibits fertilization of eggs (Brunhouse, et al., Biol. Bull., 143: 456 (1982)), formation of fertilization cone, and elongation of micro villi. (Longo, Dev. Biol., 67: 249-65 (1978); Longo, Dev. Biol., 74: 422-33 (1980); Schatten and Schatten, Dev. Biol., 78: 435-49 (1980); Byrd, J. Cell Biol., 75: 267 (1977); Eddy and Shapiro, J. Cell Biol., 71: 35-48 (1976)).
Cytochalasins also inhibit cell adhesion due to changes in the cell surface. In part these changes are due to changes in the microfilaments that affect electrical properties in the cell membrane. (Vaidyasagar, Advances in cytochalasins, Indian Drugs Res. Assoc., 149-57 (1986)). Fluctuations in these electrical properties cause morphological changes in the cell membrane. (Wadekar, et al., Exp. Cell Biol., 48: 155-66 (1980); Ghaskadbi and Mulherkar, Exp. Cell Biol., 50: 155-61 (1982)). These changes are also due in part to the action of the cytochalasins on cell surface macromolecules. This action inhibits cell growth and motility. These changes are also due to inhibition in the synthesis of mucopolysaccharide glycoprotein complex which binds cells together. (Sangar and Holtzer, Am. J. Anat., 135: 293-98 (1972); Burnside and Manasek, Dev. Biol., 27: 443-44 (1972); Brachet and Tencer, Acta Embryol. Exp., 1: 83-104 (1973)). Additionally, studies with cytochalasin H show that it produces disaggregation of cells resulting in inhibition of morphogenetic movements.
The teratogenic effects of cytochalasins have been studied in several different models, such as amphibians, mice, and chick embryo explants. In chick embryos cytochalasins have been shown to interfere with neural tube closure, cardiac looping, inhibition of primary morphogenesis of heart neural tube closure, interkinetic nuclear migration and segment formation, disaggregation of cells, microencephaly, exencephaly, and shortening of body axis. Although the exact mechanisms causing these teratogenic effects are not known, one proposed mechanism is through the inhibition of microtubules that are required for early differentiation in chick embryonic sensory neurons. (Cytochalasins: Biochemical and Cell Biological Aspect, S. W. Tannenbaum, ed., North Holland Pub. Co., 113-42 (1978); Austin, et al., Teratology, 25: 11-18 (1982); Karfunkel, J. Exp. Zool., 181: 289-302 (1972); Greenway, et al., Proc. Soc. Exp. Biol. Med., 155: 239-42 (1977); Peter, et al., Brain Res., 42 (1): 73-81 (1987); Messier and Auclair, Dev. Biol., 36: 218-23 (1974)).
Other teratogenic effects have been demonstrated in human lymphocytes, including chromosonal abnormalities. Some of the chromosomal abnormalities associated with cytochalasins include premature chromosomal condensation, extreme extension of chromosomes, ladder-like secondary constriction of chromosomes associated with bi- and multi-nucleated cells.
The cytoskeleton of the cell contains microfilaments consisting mainly of actin. Cytochalasins affect cell motility and cell shape by altering these microfilaments. In normal cells, they cause a shortening and segmentation of localized masses of actin filaments. In cell morphology studies, a generalized cell contraction was observed. Contractile proteins form actin cables condensed into masses at the base of zeiotic blebs.
Cytochalasins inhibit polymerization of actin filaments from actin nuclei by inhibiting filament elongation by blocking their growing barbed end. (Flanagan and Lin, J. Biol. Chem., 255: 835-38 (1981); Lin, et al., Biochem., Biophys. Res. Com., 122: 244-51 (1981); Casella, et al., Nature, 293: 302-5 (1981)). Several mechanisms for blocking actin filaments have been proposed. One proposed mechanism is that cytochalasins bind to the growing end of actin filaments and stimulate actin ATPase leading to depolymerization of actin filaments. (Brenner and Korn, J. Biol. Chem., 254: 9982-85 (1981)). Another proposed mechanism is that cytochalasins cut actin filaments into small pieces. (Morris and Tanenbaum, Nature, 287: 637-39 (1980)). A third proposed mechanism is that depolymerization and growth of actin filaments is inhibited by capping the filaments with cytochalasins, resulting in contraction of the cytoplasmic network and, ultimately, expulsion of the nuclei from the cell.
Cytochalasins affect the transportion of certain molecules across cell membranes. These molecules include hexose, amino acids, and various nucleosides. This transport is neither competitive nor noncompetitive (Glinsukon, et al., Toxicol. Lett., 15: 341-48 (1983); Toskulkao, et al., Nutr. Res. Int., 27: 611-18 (1983)) and is mediated by a number of functional carriers on the cell membrane. (Yamada, et al., J. Biol. Chem., 258: 9786-92 (1982)).